组蛋白乙酰化／脱乙酰化与DNA甲基化的关系

组蛋白乙酰化和脱乙酰化与DNA甲基化有密切的关系,本文介绍了DNA甲基化对组蛋白乙酰化／脱乙酰化影响,组蛋白乙酰化／脱乙酰化对DNA甲基化的影响,以及组蛋白乙酰化／脱乙酰化和DNA甲基化协同效应。     

精子发生过程中组蛋白甲基化和乙酰化

精子发生(Spermatogenesis)这一高度复杂的独特分化过程包括精原细胞发育为精母细胞、单倍体精细胞的形成和精子成熟,并以阶段特异性和睾丸特异性基因的表达、有丝分裂和减数分裂以及组蛋白向鱼精蛋白的转变为特征。表观遗传修饰在减数分裂重组、联会复合物的形成、姊妹染色体的结合、减数分裂后精子的变态、基因表达阻遏和异染色质形成过程中发挥着重要作用。其中具有一定组成形式、起抑制作用和/或激活作用的组蛋白甲基化和乙酰化标记,不仅保证了正确的染色体配对和二价染色体的成功分离,并且精确调节减数分裂特异性基因的适时表达。精子发生过程中组蛋白甲基化和/或乙酰化错误会直接影响表观遗传修饰的建立和维持,导致生精细胞异常甚至引发不育。文章旨在对精子发生过程中组蛋白甲基化和乙酰化表观遗传修饰的动态变化及其相关酶的调节机制进行综述,为进一步研究精子发生的表观遗传调控,预防男性不育疾病的发生提供基础资料。     

曲古抑菌素A对结肠癌细胞株SW480细胞周期影响的机制研究

为了研究组蛋白去乙酰化酶(HDACs)抑制剂曲古抑菌素A(TSA)对结肠癌细胞周期和凋亡的影响,初步探讨TSA作用细胞周期的可能机制,将人结肠癌细胞系SW480经TSA处理后,运用流式细胞术检测细胞周期、凋亡以及细胞周期素的变化,最后采用western-blot对细胞周期相关的基因进行检测.结果表明,TSA处理细胞后,TSA能够延缓细胞周期G1-S进程,阻滞细胞于G1期,并且影响细胞周期素cyclinE、cyclinA聚集,而对凋亡无明显的影响.Western-blot显示,TSA能够上调p21Waf1/Cip1、p27Kip1的表达,下调CDK2、cyclinE以及cycli-nA的表达.以上结果说明在结肠癌细胞中,TSA能够通过上调p21Waf1/Cip1、p27Kip1的表达以及下调CDK2、cy-clinE、cyclinA的表达,从而阻滞细胞周期于G1期,最终影响肿瘤细胞的生长,以上研究为HDAC抑制剂应用于结肠癌治疗提供了理论依据.     

曲古抑菌素A（TSA）对骨肉瘤细胞株143B增殖和凋亡的作用及其机制

观察曲古抑菌素A（TSA）对骨肉瘤细胞株143B增殖及凋亡的作用,并探讨其机制。TSA与p38抑制剂（SB203580,3μmol／L）及JNK抑制剂（SP600125,0．5μmol／L）单独或同时处理143B细胞,分别以MTT、台盼蓝染色、流式细胞术和JC—1（测定线粒体跨膜电位）法检,TSA对143B细胞的增殖、存活、周期以及凋亡的影响。应用RT-PCR、Westernblot检测Bax、Bcl．2、p38／JNK表达。结果显示,TsA能够以时间和剂量依赖方式抑制143B细胞增殖,使细胞周期阻滞于GdG。与G2／M期,并能诱导143B细胞凋亡,引起线粒体膜电位降低,促凋亡蛋白Bax表达上调,抑凋亡蛋白Bcl．2表达下调,同时使p38／ⅢK活化增加。p38／JNK4检测剂则能逆转TSA对Bax／Bcl．2的上调及抑制作用。研究结果揭示,TSA可以时间剂量依赖方式抑制143BN胞增殖,阻滞细胞周期,诱导细胞凋亡;其诱导细胞凋亡的机制可能与活化MAPK通路中p38和JNK的活性从而激发线粒体凋亡通路有关。     

DNA甲基化与组蛋白修饰对克隆胚发育的影响

在正常的受精、发育过程中,基因组DNA不对称的去甲基化、重新甲基化以及组蛋白修饰作用发生在整个受精和胚胎发育过程中。本文将从DNA甲基化、DNA不对称的去甲基化和组蛋白修饰作用就其原理,相互之间的关系及其对胚胎发育情况的影响作以综述,并对近年来,DNA甲基化与组蛋白修饰作用在胚胎发育过程中的研究作以总结。     

细菌感染对家蚕DNA甲基化与组蛋白乙酰化相关基因表达的影响

【目的】探讨DNA甲基化及组蛋白乙酰化是否参与家蚕 Bombyx mori 免疫反应的调控。【方法】对家蚕与其他生物的DNA甲基转移酶 (DNMT)、组蛋白去乙酰化酶（HDAC）与组蛋白乙酰转移酶（HAT）的蛋白序列进行系统进化分析;利用定量PCR检测家蚕5龄第3天幼虫感染病原菌绿脓杆菌 Pseudomonas aeruginosa 和金黄色葡萄球菌 Staphylococcus aureus 后 BmDNMT 1, BmHDACI-1, BmHDACI-2和 BmHAT 1在家蚕脂肪体组织中的表达变化;给家蚕5龄第2天幼虫注射DNMT, HDAC和HAT抑制剂,观察它们对家蚕感染细菌后的存活率的影响。【结果】系统进化分析显示,BmDNMT1在进化上呈现特殊性,独立于其他昆虫DNMT1的进化,而BmHDACs和BmHAT在进化上相对保守。定量PCR检测表明,在两种细菌感染后,BmDNMT1, BmHDACs 和 BmHAT1 在家蚕幼虫脂肪体中的表达水平均有不同程度的上升。然而,DNMT, HDAC和HAT抑制剂对家蚕幼虫感染细菌后的存活率并无明显影响。【结论】本研究发现感染绿脓杆菌和金黄色葡萄球菌后,家蚕幼虫脂肪体中 BmDNMT1, BmHDACs 和 BmHAT1 的表达水平有不同程度的上调,推测DNA甲基化和组蛋白乙酰化/去乙酰化可能参与家蚕免疫反应的调控。     

组蛋白甲基化与乙酰化的研究进展

组蛋白甲基化与乙酰化作为共价修饰的两种不同方式,参与许多生物学过程,并在基因表达调控中有重要作用.探讨组蛋白甲基化、乙酰化以及二者之间的关系,对认识疾病相关基因功能有重要意义,并可进一步了解基因转录的表观遗传学调控机制.     

组蛋白乙酰化与卵母细胞及胚胎的体外发育

组蛋白乙酰化及去乙酰化是表观遗传修饰一个重要部分,其对哺乳动物卵母细胞成熟和胚胎发育具有重要的调节作用。因此深入研究组蛋白乙酰化的发生机制,对于改善卵母细胞和早期胚胎的发育具有重要意义。对哺乳动物卵母细胞及胚胎发育过程中的组蛋白乙酰化动态修饰进行综述。     

牛成纤维细胞的分离与体外培养

研究了牛胎儿和成年牛皮肤组织成纤维细胞的分离、培养、纯化方法和生长特征。通过组织块贴壁培养和分离单细胞接种培养均能获得原代牛皮肤细胞。用2.5 g/L胰蛋白酶+1mmol/L EDTA和5 g/L胶原酶I联合消化牛皮肤组织较2.5 g/L胰蛋白酶+1 mmol/L EDTA消化,得到更多的单个细胞,两者之间差异极显著(P<0.01),但其死细胞比率却有较大升高;2.5 g/L胰蛋白酶+1 mmol/L EDTA消化牛胎儿组织得到的单细胞数显著高于皮肤组织消化后得到的细胞数(P<0.01),死细胞比率也高于同种酶消化的皮肤组织。分离纯化的胎儿和皮肤成纤维细胞的生长曲线都正常且相似。2.5 g/L胰蛋白酶+1 mmol/L EDTA消化贴壁细胞后死细胞率明显高于用0.5g/L胰蛋白酶+0.53 mmol/L EDTA消化的细胞(P<0.05);培养24 h后细胞贴壁率前者要明显低于后者(P<0.05)。用0.5 g/L胰蛋白酶轻度消化混杂生长的成纤维细胞和上皮样细胞,经过反复贴壁传代2～3代,可得到较纯的成纤维细胞。     

组蛋白乙酰化/去乙酰化在真核基因转录调控中的作用

真核生物中 ,染色质的基本单位是核小体。核小体由H2 A ,H2 B ,H3 ,H4构成的核心组蛋白八聚体及缠绕于其上的DNA构成。最近的研究结果表明 ,核心组蛋白的乙酰化 去乙酰化过程是调控基因活性的一个关键步骤[1] 。而含有组蛋白去乙酰化酶活性的分子有两类 :一类是与酵母RPD3同源的分子 ,另一类是与RPD3不同源的分子。它们各有其不同的来源 ,存在于各自的复合物中 ,催化不完全相同的组蛋白或其他蛋白质去乙酰化 ;这些去乙酰化酶与基因转录的调控存在着密切的关系 ,主要是介导基因转录的抑制。     

去乙酰化酶抑制剂TSA介导Ku70乙酰化对结肠癌HT29细胞凋亡和自噬的影响

目的:探讨不同浓度组蛋白去乙酰化酶抑制剂TSA对结肠癌HT29细胞的增殖、凋亡和自噬影响及其机制研究。方法:取对数生长期人结肠癌HT29细胞,采用MTT法检测不同浓度TSA处理对其细胞活力影响,并根据IC50值确定适宜给药浓度;采用流式细胞术检测不同浓度TSA处理后结肠癌HT29细胞的凋亡情况;Western blot验证空白对照组与TSA给药处理组中凋亡标志蛋白Ku70、acetrl-Ku70、Caspase3、Bax、Bcl-2和自噬标志蛋白LC3和Beclin1的表达。结果:MTT法实验结果表明TSA对结肠癌HT29细胞具有时间和浓度依赖性抑制作用,根据IC50=1.12μM,本研究中TSA的给药浓度为0.5μM和1μM;流式细胞凋亡检测结果表明TSA能够显著促进结肠癌HT29细胞凋亡,且其促凋亡作用存在浓度依赖性;此外,Western blot检测结果证实,与空白对照组相比,TSA给药处理可显著上调上述细胞中acetrl-Ku70以及促凋亡蛋白Caspase3、Bax和自噬标志蛋白LC3和Beclin1的表达,下调抗凋亡蛋白Bcl-2的表达(P<0.05)。结论:组蛋白去乙酰化酶抑制剂(TSA)的体外抗结肠癌细胞的增殖、促进细胞凋亡和自噬作用与其上调Ku70蛋白乙酰化密切相关,有望成为临床潜在抗癌靶点。     

关于组蛋白甲基化的研究

主要阐述了组蛋白甲基转移酶的类型,组蛋白H3中第9位赖氨酸甲基化与异染色质的形成、常染色体中基因表达的调控,以及与DNA甲基化之间的关系,说明了组蛋白甲基化与组蛋白乙酰化、磷酸化的相互关系, 指出组蛋白甲基化对维持细胞各种状态的平衡起到极其重要的作用。 Abstract： The types of histone methyltransferases, the relationship between methylation of Lysine 9 of H3 and the formation of heterochromatin, gene regulation in euchromatin, and that with DNA methylation, were mainly introduced. The interrelation between histone methylation and histone acetylation/phosphorylation was summarized. It is showed that histone methylation plays a very important role in maintaining the balance state of cell. The future research tendency of histone methylation was fantanstic.     

Sensitization to UV-induced apoptosis by the histone deacetylase inhibitor trichostatin A (TSA)

UV-induced apoptosis is a protective mechanism that is primarily caused by DNA damage. Cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts are the main DNA adducts triggered by UV radiation. Because the formation of DNA lesions in the chromatin is modulated by the structure of the nucleosomes, we postulated that modification of chromatin compaction could affect the formation of the lesions and consequently apoptosis. To verify this possibility we treated human colon carcinoma RKO cells with the histone deacetylase inhibitor trichostatin A (TSA) prior to exposure to UV radiation. Our data show that pre-treatment with TSA increased UV killing efficiency by more than threefold. This effect correlated with increased formation of CPDs and consequently apoptosis. On the other hand, TSA treatment after UV exposure rather than before had no more effect than UV radiation alone. This suggests that a primed (opened) chromatin status is required to sensitize the cells. Moreover, TSA sensitization to UV-induced apoptosis is p53 dependent. p53 and acetylation of the core histones may thus contribute to UV-induced apoptosis by modulating the formation of DNA lesions on chromatin.     

Dissecting the Molecular Roles of Histone Chaperones in Histone Acetylation by Type B Histone Acetyltransferases (HAT-B)

The HAT-B enzyme complex is responsible for acetylating newly synthesized histone H4 on lysines K5 and K12. HAT-B is a multisubunit complex composed of the histone acetyltransferase 1 (Hat1) catalytic subunit and the Hat2 (rbap46) histone chaperone. Hat1 is predominantly localized in the nucleus as a member of a trimeric NuB4 complex containing Hat1, Hat2, and a histone H3-H4 specific histone chaperone called Hif1 (NASP). In addition to Hif1 and Hat2, Hat1 interacts with Asf1 (anti-silencing function 1), a histone chaperone that has been reported to be involved in both replication-dependent and -independent chromatin assembly. To elucidate the molecular roles of the Hif1 and Asf1 histone chaperones in HAT-B histone binding and acetyltransferase activity, we have characterized the stoichiometry and binding mode of Hif1 and Asf1 to HAT-B and the effect of this binding on the enzymatic activity of HAT-B. We find that Hif1 and Asf1 bind through different modes and independently to HAT-B, whereby Hif1 binds directly to Hat2, and Asf1 is only capable of interactions with HAT-B through contacts with histones H3-H4. We also demonstrate that HAT-B is significantly more active against an intact H3-H4 heterodimer over a histone H4 peptide, independent of either Hif1 or Asf1 binding. Mutational studies further demonstrate that HAT-B binding to the histone tail regions is not sufficient for this enhanced activity. Based on these data, we propose a model for HAT-B/histone chaperone assembly and acetylation of H3-H4 complexes.     

Trichostatin A induces myocardial differentiation of monkey ES cells

Human embryonic stem (ES) cell lines are one of the possible sources of cardiac myocytes to be transplanted in patients with end-staged heart failure. However, prior to the application of human of ES cells for heart failure therapy, it is critical to validate their clinical use in large animals such as primates. Cynomolgus monkey ES cells have similar properties to human ES cells and can be used for primate studies. We demonstrate that 24-h stimulation by a histone deacetylase inhibitor, trichostatin A (TSA) facilitated myocardial differentiation of monkey ES cells with embryonic bodies that were seeded on gelatin-coated dishes. TSA-induced acetylating of histone-3/4 and expression of p300, one of the intrinsic histone acetyltransferases. Thus, such induction as well as inhibition of histone deacetylase may be involved in TSA-induced differentiation of cynomolgus monkey ES cells into cardiomyocytes.     

Reduced Histone H3K9 Acetylation of Clock Genes and Abnormal Glucose Metabolism in ob/ob Mice

Recent chronobiological studies found significant correlation between lack of clock function and metabolic abnormalities. We previously showed that clock gene expressions were dampened in the peripheral tissues of obese and diabetic ob/ob mice. However, the molecular mechanism of the disturbance remained to be determined. In this study, we demonstrated for the first time that acetylation levels of histone H3 lysine 9 (H3K9) at the promoter regions of clock genes, such as Dbp, Per2, and Bmal1, in the adipose tissue of ob/ob mice were significantly reduced compared with those of its control C57BL/6J mice. Treatment with histone deacetylase (HDAC) inhibitors increased Dbp, but not Per2 or Bmal1, mRNA expression in adipose tissue, and it decreased blood glucose in these animals. In addition, 2-deoxyglucose uptake activity was significantly suppressed by silencing Dbp expression in cultured adipocytes. These results suggest that reduced H3K9 acetylation and subsequent decreased mRNA expression of the Dbp gene in adipose tissue are involved in the mechanism of development of abnormal glucose metabolism in ob/ob mice. (Author correspondence: akiofuji@jichi.ac.jp)